Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Autophagy Mediates Environment-Gene Interactions in Orofacial Cleft Pathogenesis
Lucie J. Rochard, PhD1, Edward Li, BS1, Christina Nguyen, B.S.1, James F. Gusella, PhD1, Richard Maas, MD, PhD2, Cynthia Morton, PhD2, Michael Talkowski, PhD1, Eric C. Liao, MD, PhD1.
1Massachussets General Hospital, Boston, MA, USA, 2Brigham and Women's Hospital, Boston, MA, USA.

PURPOSE:
Orofacial clefts such as cleft lip and/or palate (CLP) are among the most common congenital malformations. Although CLP are associated with over 500 Mendelian syndromes, over 70% are considered nonsyndromic with no clear genetic mutations. Genome wide association studies have identified susceptibility loci for CLP, and epidemiological studies have found environmental conditions that increase its incidence. However, which mutations constitute genetic susceptibilities and how environmental stresses translate into phenotypes are largely unknown. Autophagy has gained increasing prominence as a mediator of cellular response to environmental stresses, many of which are associated with CLP. The pathway first came to our attention by analysis of balanced chromosomal translocations in CLP patients, where a break-point disrupted a critical autophagy activator ATG4C. Here, we investigate the role of autophagy in mediating environment-gene interactions during palatogenesis and CLP pathogenesis and chemical mimics of environmental stress.
METHODS:
The zebrafish model was chosen for its conservation of morphologies and molecular pathways during palatogenesis, ease of genetic and chemical manipulations, and transparent and rapidly developing embryos. Literature meta-analyses were performed for previously uncharacterized chromosomal deletions with CLP phenotypes and queried against autophagy genes. CRISPR was used to generate atg4c mutant zebrafish lines; Western blotting was used to validate the absence of Atg4c protein in mutants vs. wild type. Embryos were treated with escalating concentrations of rapamycin and deferoxamine (mimicking starvation and hypoxia by mTOR inhibition and iron chelation respectively) from 24 to 72 hours post fertilization during palatogenesis, using DMSO-treated atg4c-/- homozygotes and drug-treated atg4c+/- heterozygotes as the controls. All embryos were raised to adulthood and stained with Alcian blue for chondrocytes and examined under dissection microscopes for CLPs.
RESULTS:
Several previously uncharacterized human chromosomal deletions mapped to autophagy loci, especially ATG4C. Sequencing of a CRISPR-mediated atg4c mutant line revealed a deletion producing a truncated protein lacking its catalytic domain; Western blotting showed an absence of Atg4c protein in atg4c -/- vs. wild type. The atg4c-/- mutants were viable and exhibited normal phenotypes. However, when treated with escalating dosages of rapamycin (to inhibit mTOR pathway) and deferoxamine (to induce hypoxia), atg4c-/- embryos exhibited increasing frequencies of CLP formation compared to controls.
CONCLUSION:
The finding that atg4c-/- mutants exhibits CLP phenotype when subject to chemically induced stresses (hypoxia and starvation) supports the hypothesis that CLP pathogenesis occurs when genetic susceptibility backgrounds are modified by environmental stress to produce mutant phenotypes. This work presents the first direct evidence linking environmental stress to craniofacial malformations, and further identifies the autophagy pathway as the fundamental mechanism mediating these environment-gene interactions. Future works will seek to discover the molecular pathways upstream and downstream of autophagy during palate development and explore the potential for chemical regulators of autophagy to be used in the presence of genetic mutations and/or environmental stresses to prevent CLP formation in utero.


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